1. Field of the Invention
The present invention relates to a recombinant protein molecule, and namely a recombinant protein with modifiable binding properties to a variety of target molecules, and the recombinant protein may comprise of an antibody-like scaffold moiety wherein amino acid sequences within the scaffold are specifically or randomly altered, and random alteration of said amino acid sequences may generate a library of variant proteins where one or more proteins are capable to specifically bind to one or more target molecules, and selected variants of the recombinant protein with binding specific properties may be used as a reagent, a diagnostic or therapeutic agent.
2. General Background and State of the Art
The efficacy of a protein purification method relies on specificity of the target protein as well as efficiency both in cost and in time. The need for efficient protein purification is essential for the scientific understanding and societal applications of proteins in everyday life.
Antibodies bind in a highly specific manner to their antigen. Thus, producing an antibody which binds to a particular protein of interest is a highly sought after goal which has a wide range of applications. However, antibodies require much expense and time to produce as the production of antibodies often require immunization of animals with prepared antigens of a protein against the antigen and then isolation of the antigen specific binding antibodies. The process is costly and time consuming, and not tightly controlled as to the nature and purity of the antibody. Production of purified monoclonal antibodies, which may reduce possible artifacts in protein isolation or analysis, is far more costly and time consuming than standard polyclonal antibody production.
Recombinant antibodies can be developed from screenable libraries (e.g., phage display libraries), however, the expression of such recombinant antibodies in standard expression systems such as E. coli is problematic as yields tend to be suboptimal. As a result, alternative antibody-like scaffolds which retain the capacity to bind specifically to a target but can be highly expressed in E. coli is desirable.
The display of antibodies as antigen-binding fragments (Fabs) and single-chain variable fragments (scFvs) on filamentous phage was first described in 1990 (McCafferty et al. Nat. 348:552-54 (1990)). It provides a powerful technique for selecting a specific antibody from a mixed population of antibodies together with the gene that codes for it. The ability to co-select proteins and their genes has been exploited to enable the isolation of antigen-specific antibodies directly from repertoires or “libraries” of rearranged V-genes derived from unimmunized humans.
This ability to isolate human antibodies that bind to human proteins is of major importance for the creation of therapeutics. The problem with using murine monoclonal antibodies as therapeutics has been that they are frequently recognized as foreign by the patient's immune system. Humanizing murine antibodies can resolve the problem.
The broader the range of the library, the higher the probability of selecting a high affinity antibody to a given target. Large libraries (Vaughn et al.) are capable of generating large panels of diverse, high affinity sub-nanomolar antibodies to a given antigen. This makes it easier to obtain an antibody with the desired characteristics and is useful for both therapeutic antibodies and antibodies that will be used as research tools and reagents.
Antibodies are displayed on the surface of phage in the form of scFvs fused to the N terminus of the gIII protein. Phage with specific binding activities can then be isolated from antibody repertoires after repeated rounds of selection.
Recombinant antibodies have become an important and routinely used tool in scientific research and have also been implemented for uses in diagnostics of disease as well in various therapeutic approaches. Over 30% of biopharmaceuticals in development are recombinant antibodies of which a majority are applied towards therapies against tumor diseases and inflammation (Holliger P. et. al. Nat. Biotechnol. 23(9):1126-36 (2005), Adams G P. et. al. Nat. Biotechnol. 23(9):1147-57 (2005); Chang J T et. al. Nat. Clin. Pract. Gastro. Hepa. 4:220-8 (2006). The immunization of an animal with a specific antigen allows for the production and purification of polyclonal antibodies which can be used as detection and diagnostic reagents. However, such animal produced antibodies are limited in their use due to batch-dependence and are restricted in therapeutic application due to their immunogenicity within humans.
The generation of monoclonal antibodies through the invention of hybridoma technology helped circumvent the problems associated with polyclonal antibodies where the specificity of a particular antibody could be directed towards a desired target. The production of humanized monoclonal antibodies involves the fusion of myeloma cell lines with human B cells or transgenic murine B cells carrying a repertoire of human IgG (Lonberg N. Nat. Biotechnol. 23(9):1117(2005); Fishwild D M. et. al. Nat. Biotechnol. 14(7):845-51 (1996); Jakobovits A. Curr. Opin. Biotechnol. 695):561-6 (1995)). However, the production of monoclonal immunoglobulins from hybridomas is still dependent on in vivo methods of immunization which requires donors as well as a successful immune response. Techniques such as phage display and ribosomal display solve many of the problems associated with generating polyclonal and monoclonal antibodies as well as provide a means of improving antibodies through genetically engineering humanized versions of antibodies or fragments thereof (Hoogenbroom H R. Nat. Biotechnol. 9:1105-16 (2005); Hust M. et. al. Mol. Biol. 295:71-96 (2005)).
Unfortunately, the complex structure of antibodies poses challenges in their production. Antibodies are large protein structures that contain two light chain LC and two heavy chain HC polypeptides which are interlinked with each other in an intricate manner by numerous disulfide bridges and non-covalent interactions (Elgert K. Immunolog: Understanding the Immune System, Chapter 4:Antibody Structure and Function (1998)). Such a multifaceted protein structure requires an oxidizing environment and appropriate intracellular chaperones to assist in obtaining proper folding. Hence, cells of eukaryotic origin provide superior intracellular conditions and the protein infrastructure necessary to assist in the correct folding of antibodies.
Mammalian cells are used in the production of 60-70% of all known approved recombinant protein pharmaceuticals (Schirrmann et. al. Front. Biosci. 13:4576-94 (2008)). The advantage of using a mammalian cell line in the production of antibodies is their ability to mediate advanced protein folding and post-translational modifications. However, immunoglobulin production using mammalian cell lines is expensive. Furthermore, they raise the risk of contamination with viral pathogens or prion diseases such as bovine spongiform encephalopathy through the frequent use of undefined bovine serum in growth media. Alternatively, insect cells such as High Five or Schneider 2 cell lines are capable of complex protein folding and consequently may be used for the production of recombinant antibodies. Their disadvantage however, lies in their high cost of production, long duration before obtaining a protein product, as well as observable differences in protein glycosylation patterns (Hsu et. al. J. Biol. Chem. 272(14):9062-70 (1997)).
Yeast are an attractive alternative for the production of recombinant immunoglobulins due to their advantages of quick time of expansion, inexpensive growth conditions, can be readily altered through genetic engineering, and their capability to post-translationally modify and secrete proteins (Kim H. et. al. FEMS Yeast Res. (2014)). On the other hand, yeast may prematurely terminate transcription thus failing to express AT-rich genes (Ramanos M. et. al. Yeast 8:423-488 (1992)). Also, the propensity of yeast to hyperglycosylate heterologous proteins is problematic to producing a non-immunogenic therapeutic recombinant antibody (Sethurman N. et. al. Curr. Opin. Biotechnol. 17:341-346 (2006)).
Bacteria such as E. coli is the most common organism used for over-expressing and producing recombinant proteins. Ease and affordability of growth, rapid production of large quantities of protein, and ease of genetic manipulation make E. coli an attractive selection for the production of therapeutic recombinant immunoglobulins. Though the expression and modification of a full length immunoglobulin in a bacterial host strain is highly inefficient, smaller antibody fragments that maintain antigenic binding specificity can be readily produced in E. coli (Fellhouse F A. et. al. Making and Using Antibodies Ch. 8 CRC Press (2006)). Among the polypeptides that can be displayed on the surface of a phage library are antibodies and antibody fragments such as Fab and scFVs as described by McCafferty et. al. Nat. 348(6301):552-554 (1990); Barbas et. al. Proc. Natl. Acad. Sci. 88(18):7978-82 (1991); Burton et. al. Proc. Natl. Acad. Sci. 88(22): 10134-7 (1991); Barbas et. al. Proc. Natl. Acad. Sci. 89(10):4457-61 (1992); and Gao et. al. Proc. Natl. Acad. Sci. 96(11): 6025-30 (1999). Combining the in vitro selectivity process of a phage or ribosomal display with the production of small recombinant proteins makes E. coli a prime source for the expression of antibody like fragments. Furthermore, using synthetic DNA to introduce diversity into the antigen binding site within the antibody like proteins described herein circumvents the requirement of a natural donor.
Protein phosphorylation is an important post-translational modification that is vital for the proper function of a wide variety of proteins. Typically, a serine, threonine, or tyrosine residue within a protein may be phosphorylated which in turn may mediate a conformational change and influence the regulation of a protein's function (Johnson L N. Biochem. Soc. Trans. 37(4)627-41 (2009)). The recognition of said phosphorylated residues is also critical in relaying signaling events downstream of the effector protein. Protein domains such as Src homology-2 (SH2) and phosphotyrosine binding domains (PTB) recognize phosphotyrosine residues, whereas phosphoserine and phosphothreonine may be recognized by the 14-3-3 family of proteins, proteins that contain a tryptophan-tryptophan (WW) domain, and by the forkhead associated (FHA) domain which predominantly recognizes phosphothreonine epitopes with less specificity towards phosphoserine and phosphotyrosine (Yaffe M B Structure 7; 9(3):R33-8 (2001)).
The FHA domain is associated with proteins that are involved in diverse functions such as signal transduction cascades, gene expression and transcription, protein translocation, DNA repair, and protein degradation (Durocher D. et. al. FEBS 513:58-66 (2001)). For example, the FHA1 domain of yeast protein kinase Rad53 is involved in phospho-dependent protein:protein interactions with phosphorylated Rad9 following DNA damage and repair signaling (Durocher D. et. al. Mol. Cell 4:387-94 (1999); Lee S J. Mol. Cell Biol. 23(17):6300-14 (2003)). FHA domain containing members of the UNC104 kinesin family of proteins such as KIF1A, KIF1B, and KIF1C as well as in the KIF14 family of proteins in humans are involved in vesicular transport (Bloom G S. Curr. Opin. Cell Biol. 13:36-40 (2001); Hall D H. et. al. Cell 65:837-847 (1991); Yonekawa Y. et. al. J. cell Biol. 141:431-441 (1998); Zhao C. et. al. Cell 105:587-597 (2001)). Furthermore, FHA-containing transcription factors such as Fkh1 and Fkh2 have been identified in S. cerevisiae. Fkh1 and Fkh2 have both been shown to be master regulators of G2 transcription during yeast budding and associate with Sir2 as a means of transcription control under oxidative stress (Durocher D. et. al. FEBS 513:58-66 (2001); Linke C. et. a. Front Physiol. 4:173 (2013)).
FHA domains span approximately 100-140 amino acids in length and contain two directionally opposing β-sheets, each with five and six β strands, which fold into a β-sandwich structure that are interconnected by α-helical loops (Yaff M B. Structure 9:R33-38 (2001); Huang Y M. PlosOne 9:5 (2014)). Changes in the loop regions are the principal distinction that mediates FHA domain specificity to various target proteins (Huang Y M. PlosOne 9:5 (2014)). There are over 100 structures of FHA domains deposited in the Protein Data Bank. Protein sequence alignments of FHA domains reveal that there is a low sequence identity within the FHA family domain, however, there are five key conserved amino acid residues within the loop regions that are considered to be involved in phosphopeptide recognition (Durocher D. et. al. Mol. Cell 6:1169-82 (2000)). Although the sequences within the loop regions vary, the principle arrangement of the loop regions coordinates phosphate group binding (Huang Y M. PlosOne 9:5 (2014)).
There is currently no molecule that can support the equivalent specificity in antibody based purification and characterization techniques that is less expensive and with less delay. Furthermore, there is no molecule that is more tightly controlled for obtaining highly specific and consistent protein isolation or characterization results.